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Characterization and molecular cloning of secreted alpha-amylase with dominant activity from Mon Thong durian (Durio zibethinus Murr. cv. Mon Thong)
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Document Title
Characterization and molecular cloning of secreted alpha-amylase with dominant activity from Mon Thong durian (Durio zibethinus Murr. cv. Mon Thong)
Author
Posoongnoen S, Ubonbal R, Klaynongsruang S, Daduang J, Roytrakul S, Daduang S
Name from Authors Collection
Affiliations
Nakhon Ratchasima Rajabhat University; Khon Kaen University; Khon Kaen University; Khon Kaen University; National Science & Technology Development Agency - Thailand; Khon Kaen University
Type
Article
Source Title
REVISTA BRASILEIRA DE FRUTICULTURA
ISSN
0100-2945
Year
2021
Volume
43
Issue
3
Page
-
Open Access
gold, Green Published
Publisher
SOC BRASILEIRA FRUTICULTURA
DOI
10.1590/0100-29452021231
Format
Abstract
The secreted alpha-amylase with dominant activity was purified from the crude extract of Mon Thong durian by steps of ammonium sulphate precipitation and the affinity column chromatography. The purified alpha-amylase (DzAmy1) had a molecular mass of approximately 44 kDa. Its optimum pH and temperature for activity were 7.0 and 50 degrees C, respectively. The enzyme was stable from pH 6 to 10 and from 30 to 60 degrees C. Many metal ions did not affect amylase activity. The gene cloning of DzAmy1 was carried out and it was confirmed that DzAmy1 gene consisted of 1,254 bp open reading frame, which encoded 23 amino acids of the signal peptide and 395 amino acids of mature protein with a calculated molecular mass of 43.7 kDa. The isoelectric point of the enzyme was 5.78. DzAmy1 was shown to belong to sub-family one of the plant alpha-amylases based on phylogenctic tree analysis. Structural characterization by homology modelling suggested that it consisted of 3 domains with a catalytic triad in domain A. Recombinant DzAmy1 (rDzAmy1) was successfully expressed in Escherichia colt and had hydrolysis activity for starch and ethylidenepNP-G7, which was clearly confirmed the authenticity of DzAmy1 as a functional alpha-amylase.
Funding Sponsor
Human Resource Development in Science Project (Science Achievement Scholarship of Thailand, SAST); Khon Kaen University (KKU); Nakhon Ratchasima Rajabhat University; Research and Academic Services, KKU; Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Japan
License
CC-BY
Rights
Authors
Publication Source
WOS